The reversible binding of proteins to intracellular membrane interfaces is critical to the regulation of membrane trafficking and cell-signaling pathways. This association activates enzymes, facilitates protein-protein interactions, drives the lateral organization of protein assemblies and is responsible for the remodeling of cell membranes. It has also been shown to regulate cell-transformation and cancer. Membrane binding is often mediated by specialized protein domains that associate with the membrane interface in response to specific cellular signals; however, there is little information about the membrane interactions made by these domains. The proposed work will use an EPR based technique termed site-directed spin labeling to determine the membrane orientation and position of these domains. The forces that drive membrane attachment will also be evaluated. The C2 domains of synaptotagmin mediate calcium-dependent neuronal exocytosis, and their positions on the membrane interface in the presence of phosphatidylinositol-4,5-bisphosphate (an important signaling lipid) will be determined. Electrostatic forces may drive the assembly of signaling complexes, and the hypothesis that highly positively charged protein segments act as a scaffold to sequester proteins on the bilayer surface will be tested. The membrane position of two phosphoinositide binding domains will also be characterized and new approaches to determine protein orientation and depth on membrane surfaces evaluated. We anticipate that a better understanding of these protein-membrane interactions will lead to a better understanding of membrane trafficking and cell-signaling events. A better understanding of these events may also lead to the development of new approaches to control cell growth and cancer.